1. Field of the Invention
The present invention relates generally to biological architectural tools, and, more specifically, to the engineering of three-dimensional (3D) tissue constructs on the basis of colloidal crystals (including inverted ones) and layer-by-layer assembly to produce a scaffold for cell growth.
2. Background
3D organization of cells largely determines their function and development. Typical scaffolds for tissue cultures such as lactic acid derivatives, poly(lactide-co-glycolide), fluid microspheres, tantalum-coated carbon matrix, metallic plates, tricalcium phosphonate, hyaluronan sponges, hydroxyapatite, polyester non-woven fabric and others, can provide some degree of spatial organization of the cells. However, their micrometer scale architecture structure is not regular. In order to study the effects of 3D cell contacts one needs a scaffold with distinct order. Well-organized structure of the scaffolds can help to more accurately determine and realize in practice the optimal number and modality of intercell contacts, which is critical for adequate tissue development. Additionally, the uniformity of nutrient fluxes present in ordered systems will be beneficial for achieving homogeneity of cells developing on the scaffolds including stem cell differentiation.
Therefore, it is important to find a method of preparation of cell scaffolds providing 3D crystallinity in the micrometer to millimeter scale as well as space sufficient to accommodate cells, which be achieved in direct and inverted colloidal crystals. Essentially, colloidal crystals are hexagonally ordered lattices of spherical particles with a diameter from several nm to several millimeters. As is well known in the art, the colloidal crystals can be easily self-assembled by sedimentation and then annealed to form solids. Sedimentation of colloidal particles has traditionally been conducted very slowly to organize colloidal crystals into a 3D structure. Other problems known for currently used scaffolds include pH changes during biodegradation which can negatively affect the cell development, biocompatibility with some cell cultures, insufficient cell adhesion and others related to the composition and structure of the scaffold surface. These problems may be substantially alleviated or eliminated by taking advantage of the new surface modification procedure known as layer-by-layer assembly. Due to universality of this technique it can also be applied to achieve desirable surface set of properties and activities for different cell cultures and on different scaffolds. As well, a versatile surface modification procedure can optimization of nutrient delivery and to control the release of differentiation factors and attachment of cells to the scaffold.
“Layer-by-layer” (LBL) is a term used to describe a film deposition process that has been applied for oppositely charged polyelectrolytes. The LBL film deposition process has also been extended to the layer-by-layer assembly of nanoparticle colloids. LBL procedure involves sequentially dipping a substrate into solutions of oppositely charged species alternating with water rinse. In each dipping cycle, a layer of the species, preferably a monolayer or a nanolayer, adsorbs to the substrate. The rinse step removes excess species material. Subsequent dipping cycles result in enhanced adsorption of an oppositely charged species, which is also accompanied by a switch in surface charge. The surface charge switch promotes the adsorption of a following layer. This cycle can be repeated as many times as needed to build up a multilayer of desirable thickness.
One of the major driving forces of LBL is the electrostatic attraction between positive and negative charges located on a solid surface and polyelectrolytes, colloids and other species in solution. Important thermodynamic contributions to film stability are also made by van der Waals interactions. Alternation of layers of positively and negatively charged components is a key principle of the layer-by-layer assembly. The monomolecular nature of layers deposited in each cycle of the LBL technique affords nm scale precision in thin film thickness.
Since the LBL method is quite simple and effective, it has been applied to a variety of charged species from classical inorganic colloids to DNAs. Importantly, assembled biopolymers retain their 3D structure and biological activity. This property of assembled biopolymers has been utilized for enhancement of biocompatibility and attachment of living cells to nanostructured composites.
Overall, there is a recognized need in the field of tissue engineering for well ordered 3D tissue constructs with tunable surface properties. It is thus an object of the present invention to provide methods for engineering improved scaffolds for cell growth.